Enkephalinase inhibitors

ABSTRACT

The degradation of enkephalins in a mammalian host is inhibited by administration of an enkephalinase enzyme inhibitor of the formula ##STR1##

This is a division of application Ser. No. 310,192, filed Oct. 9, 1981now U.S. Pat. No. 4,401,677.

BACKGROUND OF THE INVENTION

Roques et al. (Nature Vol. 288, November 1980, p. 286-288) disclose thatthiorphan, (DL-3-mercapto-2-benzylpropanoyl)-glycine, is an inhibitor ofenkephalinase in vitro in nanomolar concentration and in vivo aftereither intracerebroventricular or systemic administration.

Ondetti et al. in U.S. Pat. No. 4,053,651 discloses that variousmercaptoalkanoyl amino acids are useful hypotensive agents due to theirability to inhibit the angiotensin converting enzyme.

Sundeen et al. in U.S. Pat. No. 4,235,885 disclose that mercaptoalkanoylamino acids of the formula ##STR2## wherein R₂ can be an amino acid areuseful in the treatment of rheumatoid arthritis due to their mammaliancollagenase inhibitory activity.

BRIEF DESCRIPTION OF THE INVENTION

This invention is directed to the discovery that enkephalinase isinhibited by compounds of the formula ##STR3## and salts thereof. Informula I, and throughout the specification, the symbols are as definedbelow.

R₁ is straight or branched chain alkyl of 1 to 4 carbons, benzyl, orphenethyl.

R₂ is straight or branched chain alkyl of 1 to 4 carbons. ##STR4##--(CH₂)_(n) --S-alkyl wherein alkyl is straight or branched chain of 1to 4 carbons, ##STR5##

n is an integer from 1 to 4.

Preferred compounds of formula I to be employed within the method ofthis invention are those wherein: ##STR6##

DETAILED DESCRIPTION OF THE INVENTION

As taught by Ondetti et al. and Sundeen et al. in the above notedpatents, the compounds of formula I can be prepared by acylating anamino acid of the formula ##STR7## with an acid or its chemicalequivalent of the formula ##STR8## wherein R₃ is a protecting group suchas lower alkanoyl of 1 to 4 carbons, preferably acetyl, or benzoyl. Theabove acylation yields the intermediate of the formula ##STR9##Treatment of the intermediate of formula IV by conventional hydrolysisor ammonolysis yields the mercapto compounds of formula I.

This acylation reaction can be effected in the presence of a couplingagent such as dicyclohexylcarbodiimide or the like, or the acid offormula III can be activated by formation of its mixed anhydride,symmetrical anhydride, acid halide, active ester or the use of Woodwardreagent, K, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline or the like.For a review of the methods of acylation, see Methoden der OrganischenChemie (Houben-Weyl), Vol. XV, part II, page 1 et seq. (1974).Preferably, the acid halide, especially the acid chloride, of formulaIII is reacted with the amino acid or its hydrochloride salt of formulaII.

The intermediates of formula IV can also be prepared by reacting theamino acid of formula II or an ester thereof with an acrylic acid or itschemical equivalent of the formula ##STR10## to yield, after removal ofthe ester group, the intermediate of the formula ##STR11## Preferably,the acrylic acid of formula V is converted to an activated form such asthe acid chloride by treatment with thionyl chloride.

The intermediate of formula VI is then treated with a thio acid of theformula

    R.sub.3 --SH                                               (VII)

to yield the intermediate of formula IV.

The compounds of formula I contain two asymmetric carbon atoms. As shownin the formulas, the asymmetric carbon in the amino acid portion of themolecule is in the L-configuration. The asymmetric carbon in themercapalkanoyl sidechain can be in the D-, L-, or D,L-configuration. Thecompounds of formula I accordingly exist in stereomeric forms or asracemic mixtures thereof. All of these forms can be utilized in themethod of this invention. The above described synthesis can utilize thestarting compounds in the form of a racemic mixture or as a stereomer.

The compounds of formula I form basic salts with a variety of inorganicor organic bases. The pharmaceutically acceptable salts of the compoundsof formula I are useful within the method of this invention. Suchpharmaceutically acceptable salts include alkali metal salts such assodium or potassium, alkaline earth metal salts such as calcium ormagnesium, and salts derived from amino acids like arginine, lysine,etc. The salts are produced by reacting the acid form of the compoundwith an equivalent of the base supplying the desired basis ion in amedium in which the salt precipitates or in aqueous medium and thenlyophilizing.

The compounds of formula I when administered to a mammalian specie areuseful analgesic agents due to their enkephalinase inhibition activity.While not limiting the scope of this invention to a specific theory ormechanism of action, it has been suggested that the endogenous opiatepentapeptides, [Met⁵ ]-enkephalin(Tyr-Gly-Gly-Phe-Met) and [Leu⁵]-enkephalin(Try-Gly-Gly-Phe-Leu), are neurotransmitters involved incentral pain mediation (Hughes, et al., Nature, Vol. 258, December 1975,p. 577-579) and that these endogenous opiate peptides are functionallyinactivated by cleavage of their Gly³ -Phe⁴ peptide bonds by a specificpeptidyldipeptide hydrolase, enkephalinase, presumed to be specificallylocated at nerve terminals in the brain where enkephalins are released(Malfroy, et al., Nature, Vol. 276, November 1978, p. 523-526). Specificinhibitors of this enkephalinase enhance the release of endogenousenkephalins from isolated brain slices (Patey, et al., Science, Vol.212, June 1981, p. 1153-1155) and cause analgesia in mice that isreversed by the opiate anatogonist naloxone (Roques, et al., supra). Inaddition to analgesia, other pharmacological actions such as antitussiveor antidiaharreal activities may result from prolonging the action ofthe body's natural opiates released from peripheral as well as centralsites.

Thus, by the administration of a composition containing one or acombination of compounds of formula I or a pharmaceutically acceptablesalt thereof, pain is alleviated in the mammalian host. A single dose,or preferably two to four divided daily doses, provided on a basis ofabout 0.1 to about 100 mg. per kilogram of body weight per day,preferably about 1 to about 50 mg. per kilogram per day, produces thedesired analgesic activity. The composition is preferably administeredorally but parenteral routes such as subcutaneous can also be employed.

The compounds of formula I can be utilized as enkephalinase inhibitorsfor the alleviation of pain by formulating in compositions such astablets, capsules, or elixirs for oral administration or in sterilesolutions or suspensions for parenteral administration. About 10 to 500mg. of a compound or mixture of compounds of formula I orphysiologically acceptable salt is compounded with a physiologicallyacceptable vehicle, carrier, excipient, binder, preservative,stabilizer, flavor, etc., in a unit dosage form as called for byaccepted pharmaceutical practice. The amount of active substance inthese compositions or preparations is such that a suitable dosage in therange indicated is obtained.

Illustrative of the adjuvants which may be incorporated in tablets,capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid and the like; a lubricant such as magnesiumstearate; a sweetening agent such as sucrose, lactose or saccharin; aflavoring agent such as peppermint, oil of wintergreen or cherry. Whenthe dosage unit form is a capsule, it may contain in addition tomaterials of the above type a liquid carrier such as a fatty oil.Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice by dissolving or suspending theactive substance in a vehicle such as water for injection, a naturallyoccurring vegatable oil like sesame oil, coconut oil, peanut oil,cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate orthe like. Buffers, preservatives, antioxidants and the like can beincorporated as required.

The following examples are illustrative of the invention and constituteespecially preferred embodiments. All temperatures are in degreescelsius.

EXAMPLE 1 N-(DL-3-Mercapto-2-methyl-1-oxopropyl)-L-arginine

This compound is prepared as set forth in Examples 20 and 21 of U.S.Pat. No. 4,053,651 of Ondetti et al.

(a) N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-L-arginine

L-Arginine (2.61 g.) and sodium carbonate (800 mg.) are dissolved in 20ml. of water and stirred in an ice bath. To this mixture, sodiumcarbonate (2.4 g.) in 10 ml. of water is added followed immediately byD,L-3-acetylthio-2-methylpropanoyl chloride which is washed in with 5ml. of ether. The pH of the reaction mixture is about 8. The ice bath isremoved and the reaction mixture is stirred for 1.5 hours at roomtemperature. The reaction mixture is neutralized with 50 ml. ofAG-50W-X2 resin and applied to an 80 ml. column of the same. The columnis washed with water until the eluent is no longer acidic to pH paperand then with buffer pH 6.15 (900 ml. of water, 100 ml. of pyridine, 4ml. of acetic acid). The product containing fractions are pooled andlyophilized to yield 4.1 g. of crude material. Recrystallization frommethanol/either yields 3.86 g. of N-(DL-3-acetylthio-2-methyl-1-oxopropyl)-L-arginine; m.p. 133°.

(b) N-(DL-3-Mercapto-2-methyl-1-oxopropyl)-L-arginine

N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-L-arginine (1 g.) is dissolvedin a mixture of water (5 ml.) and concentrated ammonia (5 ml.). Afterone hour at room temperature, the pH is adjusted to 6.6 withconcentrated hydrochloric acid while chilling in an ice bath. Thesuspension is applied to a 30 ml. column of AG-50W-X2 ion exchangeresin. The column is eluted with water until no longer acidic to pHpaper and then with pyridine-acetate buffer pH 6.5. The fractionscontaining the desired product are pooled, concentrated to dryness andlyophilized to yield 866 mg. ofN-(DL-3-mercapto-2-methyl-1-oxopropyl)-L-arginine; m.p. 100°.

Anal. calc'd. for C₁₀ H₂₀ N₄ O₃ S: C, 44.40; H, 7.20; N, 20.23; S,11.29. Found: C, 44.65; H, 7.58; N, 19.99; S, 10.95.

EXAMPLE 2 N-[DL-2-(Mercaptomethyl)-1-oxo-3-phenylpropyl]-L-arginine

This compound is prepared as set forth in Example 34 of Ondetti et al.U.S. Pat. No. 4,053,651.

(a) N-[DL-2-(Acetylthiomethyl)-1-oxo-3-phenylpropyl]-L-arginine

L-Arginine hydrochloride (1.85 g.) and 940 mg. of sodium carbonate aredissolved in 12 ml. of water and stirred in an ice bath. To thismixture, sodium carbonate (1.4 g.) in 6 ml. of water is added followedimmediately by DL-3-acetylthio-2-benzylpropanoic acid chloride (2.25 g.,Example 32 of 4,053,651) and washed in with 5 ml. of ether. The ice bathis removed and the pH is about 8.0. The reaction is kept at roomtemperature for 1.5 hours and after 30 minutes a precipitate develops.Ion exchange resin AG-50W-X2 is added until the mixture is acidic andthe suspension is then applied to a 50 ml. AG-50W-X2 column. The columnis washed with water until no longer acidic to pH paper and then elutedwith pyridine-acetate buffer pH 6.5. The product containing fractionsare pooled, concentrated to dryness, removed from benzene and absoluteethanol and dried in vacuo. The crude product is triturated with etherand filtered to yield 2.12 g. ofN-[DL-2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-L-arginine.

(b) N-[DL-2-(Mercaptomethyl)-1-oxo-3-phenylpropyl]-L-arginine

N-[DL-2-(Acetylthiomethyl)-1-oxo-3-phenylpropyl]-L-arginine (1 g.) istreated with a cold solution of 2.2 ml. of water and 1.6 ml. ofconcentrated ammonia for one hour at room temperature. Sufficient ionexchange resin AG-50W-X2 is added to adjust the pH to 5-6 and thesuspension is added to a 50 ml. AG-50W-X2 column. The column is washedwith water until the eluent is no longer acidic. The column is theneluted with pyridine-acetate buffer pH 6.5 and a mixture of this bufferpH 6.5-methanol (8:2). The product containing fractions are pooled,concentrated to dryness and removed once from absolute ethanol. Thecrude product (403 mg.) is precipitated from methanol/ether to yield 292mg. of N-[DL-2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-L-arginine; m.p.135°.

Anal. cal'd. for C₁₈ H₂₄ N₄ O₃ S.H₂ O: C, 52.00; H, 7.10; N, 15.10; S,8.65. Found: C, 52.50; H, 6.94; N, 14.97; S, 8.34.

EXAMPLE 3 N-(DL-3-Mercapto-2-methyl-1-oxopropyl)-L-tryptophan (a)N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-L-tryptophan

L-Tryptophan (3.06 g.) is dissolved in 17.6 ml. of 0.85N sodiumhydroxide with stirring in an ice bath. To this solution, 7.5 ml. of 2Nsodium hydroxide is added followed immediately byDL-3-acetylthio-2-methylpropanoyl chloride (2.7 g.) and 5 ml. of ether.The ice bath is removed and the pH of the reaction mixture is between7-8. After two hours, the reaction mixture is extracted twice with ethylacetate, the aqueous phase is acidified with concentrated hydrochloricacid, saturated with sodium chloride and extracted with ethyl acetate toyield 4.9 g. of crude product. Purification is achieved on a 150 g.silica gel column eluting with benzene/acetic acid (7:2) to yield 2.3 g.of N-(DL-3-acetylthio-2-methyl-1-oxopropyl)-L-tryptophan.

(b) N-(DL-3-Mercapto-2-methyl-1-oxopropyl)-L-tryptophan

N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-L-tryptophan (918 mg.) is takenup in 9 ml. of methanol with stirring under an argon blanket.Concentrated NH₄ OH (9 ml.) is added. After 30 minutes, the methanol isremoved in vacuo, the aqueous phase is acidified with solid potassiumbisulfate and extracted into ethyl acetate to yield 790 mg. ofN-(DL-3-mercapto-2-methyl-1-oxopropyl)-L-tryptophan.

Anal. calc'd. for C₁₅ H₁₃ N₂ O₃ S.H₂ O: C, 55.60; H, 4.76; N, 8.65.Found: C, 56.37; H, 5.56; N, 8.81.

EXAMPLE 4 N-(DL-3-Mercapto-2-methyl-1-oxopropyl)-L-tyrosine (a)N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-L-tyrosine

L-Tyrosine (2.718 g.) is suspended in 17.6 ml. of 0.85N sodium hydroxidewith stirring in an ice bath. To this mixture, 7.5 ml. of 2N sodiumhydroxide is added followed immediately by 2.7 g. ofDL-3-acetylthio-2-methylpropanoyl chloride and 5 ml. of ether. After 15minutes a precipitate forms. After 1.5 hours, 30 ml. of water are added.After 4 hours, the mixture is diluted to 300 ml. with water andextracted twice with ethyl acetate. The aqueous phase is acidified withconcentrated hydrochloric acid and thoroughly extracted into ethylacetate. The crude product (4 g.) is chromatographed on silica geleluting with benzene/acetic acid (7:3) to give 1.6 g. ofN-(DL-3-acetylthio-2-methyl-1-oxopropyl)-L-tyrosine.

(b) N-(DL-3-Mercapto-2-methyl-1-oxopropyl)-L-tyrosine

N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-L-tyrosine (1.1 g.) is taken upin methanol with stirring under an argon blanket and then treated with10 ml. of concentrated NH₄ OH. After 30 minutes, the methanol is removedin vacuo. The aqueous phase is taken up in ethyl acetate and acidifiedwith 10% potassium bisulfate. The ethyl acetate mixture is washed withwater, dried over MgSO₄ and concentrated to dryness in vacuo.Purification is achieved by silica gel column chromatography elutingwith benzene/acetic acid (7:3) to yield 394 mg. ofN-(DL-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine.

EXAMPLE 5 3-Hydroxy-N-(DL-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine(a) N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-3-hydroxy-L-tyrosine

3,4-Dihydroxy-L-phenylalanine (3.94 g.) is taken up in 23.6 ml. of 0.85Nsodium hydroxide with stirring in an ice bath under a blanket of argon.To this mixture, 10 ml. of 2N sodium hydroxide is added followedimmediately by 3.6 g. of DL-3-acetylthio-2-methylpropanoyl chloride in10 ml. of ether. The ice bath is removed. After 45 minutes a precipitatecomes out of solution and 30 ml. of water is added. The precipitae doesnot become soluble. After four hours, the reaction is acidified withconcentrated hydrochloric acid while chilling and then extracted intoethyl acetate to yield 5.8 g. of crudeN-(DL-3-acetylthio-2-methyl-1-oxopropyl)-3-hydroxy-L-tyrosine.

(b) 3-Hydroxy-N-(DL-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine

N-(DL-3-Acetylthio-2-methyl-1-oxopropyl)-3-hydroxy-L-tyrosine (4.9 g.)is suspended in 20 ml. of water in an ice bath under a blanket of argon.To this suspension, 20 ml. of concentrated NH₄ OH is added. Thesubstrate goes into solution within four minutes. After thirty minutesat room temperature the reaction is chilled, acidified with concentratedhydrochloric acid, and extracted into ethyl acetate to yield 4.4 g. ofcrude product. This material is taken up in chloroform/acetic acid (7:3)and applied to 150 g. column of silica gel wrapped in aluminum foil andthe fractions are worked up in a darkened laboratory. Elution isperformed with chloroform/acetic acid (7:3) to yield 2.9 g. of3-hydroxy-N-(DL-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine. Atert-butylamine salt is formed in ethyl acetate on a small aliquot.

Anal. calc'd. for C₁₃ H₁₇ NO₅ S.NH₂ C(CH₃)₃. 1.6H₂ O: C, 50.87; H, 7.38;N, 6.98; S, 7.98. Found: C, 50.94; H, 7.54; N, 6.96; S, 7.66.

EXAMPLE 6 3-Hydroxy-N-(D-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine (a)N-(D-3-Acetylthio-2-methyl-1-oxopropyl)-3-hydroxy-L-tyrosine

D-3-Acetylthio-2-methylpropanoyl chloride (10.84 g.) is added in fiveportions to 3,4-dihydroxy-L-phenylalanine (11.83 g.) simultaneously withequal portions of 60 ml. 1N sodium hydroxide at 0° under a nitrogenatmosphere. The additions require about one hour and the final pH is9.0. The reaction is allowed to stir at room temperature for 4 hours. Aclear solution is not obtained. The solution is layered with 200 ml. ofethyl acetate and made strongly acid with concentrated hydrochloricacid. The aqueous extract is then extracted twice with 200 ml. of ethylacetate and the combined organic layers are dried over MgSO₄. Thesolvent is removed to yield 16 g. of crude product in the form of atacky residue. The 16 g. residue is dissolved in 30 ml. of ethylacetate, 250 ml. of ether are added and then 9.05 g. ofdicyclohexylamine in 30 ml. of ether. A white crystalline solid formsimmediately and is filtered and dried overnight to yield 14 g. ofN-(D-3-acetylthio-2-methyl-1-oxopropyl)-3-hydroxy-L-tyrosine,dicyclohexylamine salt; m.p. 75°-80° (soft at 60°).

Anal. Calc'd. for C₁₅ H₁₉ NO₆ S.C₁₂ H₂₁ N.2H₂ O: C, 58.07; H, 7.94; N,5.02. Found: C, 58.29; H, 7.17; N, 4.81.

14 g. of the above dicyclohexylamine salt is converted to the free acidusing ethyl acetate and potassium bisulfate. Concentration of the driedethyl acetate solution yields 12 g. ofN-(D-3-acetylthio-2-methyl-1-oxopropyl)-3-hydroxy-L-tyrosine.

(b) 3-Hydroxy-N-(D-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine

N-(D-3-Acetylthio-2-methyl-1-oxopropyl)-3-hydroxy-L-tyrosine (12.2 g.)gradually goes into solution in 60 ml. of water (ice bath and nitrogenatmosphere) and the solution is treated with 60 ml of concentrated NH₄OH and stirred at room temperature under nitrogen for one hour. Thesolution is then layered with ethyl acetate and made strongly acidicwith concentrated hydrochloric acid. The acid aqueous layer is extractedtwice with 200 ml. of ethyl acetate and the combined organic layers aredried over MgSO₄. The solvent is removed to yield 7.5 g. of a brittlefoam after drying overnight. The 7.5 g. is dissolved in 150 ml. ofwater, filtered and lyophilized to yield 7 g. of3-hydroxy-N-(D-3-mercapto-2-methyl-1-oxo-propyl)-L-tyrosine as ahygroscopic solid; m.p. 50°-55° (soft at 45°).

Anal. Calc'd for C₁₃ H₁₇ NO₅ S.0.5H₂ O: C, 50.63; H, 5.88; N, 4.54; S,10.39. Found: C, 50.09; H, 6.06; N, 4.60; S, 10.35.

EXAMPLE 7 (±)-N² -[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]-L-arginine(a) (±)-N² -[2-(Acetylthiomethyl)-4-methyl-1-oxopentyl]-L-arginine

L-Arginine (1.9 g., 0.11 mole) is dissolved in 10 ml. of watercontaining sodium bicarbonate (0.9 g., 0.11 mole). This solution iscooled to 5° and 2-(acetylthiomethyl)-4-methylpentanoyl chloride (2.5g., 0.11 mole) [prepared as set forth by Sundeen et al. in Example 2(a)of U.S. Pat. No. 4,235,885] in 5 ml. of ether is added dropwise. The pHof the reaction mixture is maintained between 7-8 by the occasionaldropwise addition of saturated aqueous sodium bicarbonate. Afterstirring at room temperature for 5 hours, the reaction mixture is washedwith ether and the aqueous solution is lyophilized overnight to yield3.6 g. of crude product. This material is dissolved in 10 ml. ofabsolute ethanol and poured through a 100 ml. silica gel column(previously washed with ethanol). After 200 ml. of ethanol go throughthe column, product (2.3 g.) is eluted in two 50 ml. fractions. Thismaterial is chromatographed through 100 g. of Avicel using (9:1)methanol:water. Product is eluted in two 100 ml. fractions and the firstfraction is lyophilized to yield 0.6 g. of analytically pure (±)-N²-[2-(acetylthiomethyl)-4-methyl-1-oxopentyl]-L-arginine; softens at64°-80°.

Anal. calc'd. for C₁₅ H₂₈ N₄ O₄ S.0.75 H₂ O: C, 48.18; H, 7.95; N,14.98; S, 8.52. Found: C, 48.14; H, 7.78; N, 14.69; S, 8.58.

(b) (±)-[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]-L-arginine

(±)-N² -[2-(Acetylthiomethyl)-4-methyl-1-oxopentyl]-L-arginine (0.6 g.)is dissolved in 5 ml. of water and purged with argon. To this solution,2 ml. of 37% aqueous NH₄ OH is added and stirred at room temperature for2 hours. It is lyophilized overnight and the resulting white solid iswashed with 60 ml. of acetonitrile containing 6 drops of water. Thegranular solid is filtered and dried in vacuo at 60° for 2 hours toyield (±)-N² -[2-(mercaptomethyl)-4-methyl-1-oxopentyl]-L-arginine,softens at 127°.

Anal. calc'd. for C₁₃ H₂₆ N₄ O₃ S.H₂ O: C, 46.41; H, 8.39; N, 16.65; S,9.53. Found: C, 46.38; H, 8.21; N, 16.38; S, 9.42.

EXAMPLE 8 (±)-N-[2-Mercaptomethyl)-1-oxo-3-phenylpropyl]-L-leucine,ammonium salt (a) 2-Phenylmethyl-2-propenoyl chloride

Benzyl malonic acid (13 g., 0.067 mole) is mixed with 40% aqueousdimethylamine (7.6 g., 0.068 mole) and 37% formalin (5.4 g., 0.068 mole)in 150 ml. of water. The voluminous solid which forms in 15 minutes isfiltered after two hours, washed with water and dried partially in airto give 20.8 g. of benzyl malonic acid dimethylamine.

This solid is melted in a 170° oil bath and heated for 10 minutes untilamine evolution stops and bubbling virtually ceases. The cooled product,a mobile liquid, is acidified with 10% potassium bisulfate, extractedwith hexane, dried (Na₂ SO₄) and evaporated to give 6.3 g. of solid2-benzylacrylic acid.

The 2-benzylacrylic acid (6.0 g.) is dissolved in ether and 10 ml. ofthionyl chloride are added dropwise. Reaction temperature is allowed torise to 35°. After stirring for 2 hours, the mixture is concentrated invacuo to yield 6.4 g. of crude 2-phenylmethyl-2-propenoyl chloride.

(b) N-(2-Methylene-1-oxo-3-phenylpropyl)-L-leucine, methyl ester

L-Leucine, methyl ester, hydrochloride (2.72 g.) is dissolved in 70 ml.of dichloromethane. Triethylamine (3.03 g.) is added, followed by thedropwise addition of 2-phenylmethyl-2-propenoyl chloride (6.4 g.) at 0°.After the reaction is stirred for 3 hours, it is diluted with 100 ml. ofdichloromethane and washed sequentially with 10% aqueous hydrochloricacid, aqueous sodium bicarbonate, and water. the organic phase is dried(MgSO₄), filtered, and concentrated in vacuo to yield 4.4 g. ofN-(2-methylene-1-oxo-3-phenylpropyl)-L-leucine, methyl ester; m.p.67°-71°.

(c) N-(2-Methylene-1-oxo-3-phenylpropyl)-L-leucine

The methyl ester product from part (b) is dissolved in 150 ml. ofmethanol. Sodium hydroxide (0.8 g.) in 150 ml. of water is addeddropwise and the reaction mixture is heated at 60° for 30 minutes. Thereaction mixture is concentrated in vacuo to remove the methanol andthen diluted with 50 ml. more of water. The aqueous phase is washed withether and then acidified with 10% aqueous hydrochloric acid. The productis extracted with two 200 ml. portions of dichloromethane, dried(MgSO₄), filtered, and concentrated in vacuo to yield 4.2 g. ofN-(2-methylene-1-oxo-3-phenylpropyl)-L-leucine; m.p. 56°-60°.

(d) (±)-N-[2-(Acethylthiomethyl)-1-oxo-3-phenylpropyl]-L-leucine

The N-(2-methylene-1-oxo-3-phenylpropyl)-L-leucine (4.2 g.) is dissolvedin 50 ml. of chloroform and 5 ml. of thiol acetic acid. This mixture isstirred under nitrogen overnight and then concentrated in vacuo to yieldan almost colorless oil which slowly crystallizes out of ether to yield2.4 g of (±)-N-[2-(acethylthiomethyl-1-oxo-3-phenylpropyl]-L-leucine;m.p. 90°-92°.

Anal. calc'd. for C₁₈ H₂₄ NSO₄ : C, 61.51; H, 7.17; N, 3.99; S, 9.12.Found: C, 61.50; H, 7.21; N, 4.02; S, 9.06.

(e) (±)-N-[2-(Mercaptomethyl)-1-oxo-3-phenylpropyl]-L-leucine, ammoniumsalt

(±)-N-[2-(acetylthiomethyl)-1-oxo-3-phenylpropyl]-L-leucine (1.0 g.) issuspended in 50 ml. of distilled water which is purged with argon. Tothis suspension is added 2 ml. of 37% NH₄ OH and the reaction mixture isstirred under argon for 2 hours. It is lyophilized overnight to yield awhite solid. This solid is stirred with 50 ml. of acetonitrile for 2hours, filtered, and dried in vacuo for 12 hours to give 0.7 g. of(±)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-L-leucine, ammoniumsalt; m.p. 102°-115° with evolution of ammonia.

Anal. calc'd. for C₁₆ H₂₆ N₂ O₃ S: C, 58.87; H, 8.03; N, 8.58; S, 9.82.Found: C, 58.70; H, 7.96; N, 8.24; S, 9.47.

EXAMPLES 9-22

Following the procedure of Examples 1 to 7 but employing the acidchloride shown in Col. I and the amino acid shown in Col. II theintermediate shown in Col. III is obtained. Removal of the R₃ protectinggroup yields the compound shown in Col IV which is useful in the methodof treatment of this invention.

    ______________________________________                                        Col. I               Col. II                                                   ##STR12##                                                                                          ##STR13##                                               Col. III                                                                       ##STR14##                                                                    Col. IV                                                                        ##STR15##                                                                    Ex-                                                                           ample R.sub.3    R.sub.2     R.sub.1                                          ______________________________________                                         9                                                                                   ##STR16##                                                                                ##STR17##                                                                                 ##STR18##                                       10                                                                                   ##STR19##                                                                                ##STR20##                                                                                 ##STR21##                                       11                                                                                   ##STR22##                                                                                ##STR23##  CH.sub.3                                         12                                                                                   ##STR24##                                                                                ##STR25##                                                                                 ##STR26##                                       13                                                                                   ##STR27##                                                                                ##STR28##                                                                                 ##STR29##                                       14                                                                                   ##STR30##                                                                                ##STR31##                                                                                 ##STR32##                                       15                                                                                   ##STR33##                                                                                ##STR34##  CH.sub.2 SH                                      16                                                                                   ##STR35##                                                                                ##STR36##  (CH.sub.2).sub.2SCH.sub.3                        17                                                                                   ##STR37##                                                                                ##STR38##  (CH.sub.2).sub.4NH.sub.2                         18                                                                                   ##STR39##                                                                                ##STR40##                                                                                 ##STR41##                                       19                                                                                   ##STR42## H.sub.5 C.sub.2                                                                            ##STR43##                                       20                                                                                   ##STR44##                                                                                ##STR45##                                                                                 ##STR46##                                       21                                                                                   ##STR47## H.sub.3 C(CH.sub.2).sub.2                                                                  ##STR48##                                       22                                                                                   ##STR49## H.sub.3 C   CH.sub.2CH(CH.sub.3).sub.2                       ______________________________________                                    

EXAMPLE 23

The following experiment demonstrated the ability of the compounds offormula I to inhibit the cleavage of [³ H-Tyr¹, Leu⁵ ]-enkephalin byenkephalinase purified from membrane fractions of sheep corpus striatum.

Frozen sheep striatia were homogenized in 20 volumes of 50 mM Tris(i.e., 2-amino-2-hydroxymethyl-1,3-propanediol)-hydrochloric acidbuffer, pH 7.7, and centrifuged at 37,000×g. for 15 minutes. Thesedimented material (membrane fraction), after being washed three timesby resuspension and recentrifugation, was solubilized by stirring withhalf the original volume of 50 mM Tris-hydrochloric acid buffer, pH 7.7,containing 1% Triton X-100 (i.e., polyethylene glycol p-isooctylphenylether) nonionic detergent, followed by centrifugation for 2 hours at37,000×g. The supernatant solution (solubilized enzyme) was applied to a1.5×30 cm--column of Whatman DE52--cellulose, previously equilibratedwith the same buffer-detergent mixture. Protein was eluted from thecolumn with a one liter gradient of 0-0.4M sodium chloride in the samebuffer mixture, with 8.7 ml. fractions being collected. Enkephalinaseactivity in fractions 10 to 17 was pooled, concentrated, and applied toa 2.5×80 cm. column of Sephadex G-200 equilibrated with 50 mMTris-hydrochloric acid, pH 7.0, containing 1% polysorbate 80 detergent.The enkephalinase activity emerging as a single peak from Sephadex G-200was used to test for inhibition activity.

Activity of the enzyme for purification and inhibitor studies wasdetermined by the following radiochromatographic method. An assayincubation mixture of 0.02 ml. containing 70 nM [³ H-Tyr¹, Leu⁵]-enkephalin (50,000 cpm), an appropriate dilution of the enzyme, andthe inhibitor in a final concentration of 125 mM Tris-hydrochloric acid,pH 7.0, was incubated for 15 minutes at 37° before stopping theenzymatic reaction with 0.005 ml. of 1N hydrochloric acid. An aliquot of0.01 ml. of acidified reaction mixture was spotted onto a Whatman LK 50silica gel thin-layer chromatography plate along with the appropriatepeptide standards. After development of the plate in a solvent composedof isopropanol: ethyl acetate: 5% acetic acid (2:2:1), the spotscorresponding to Tyr, Tyr-Gly, Tyr-Gly-Gly, and unreacted enkephalin(Try-Gly-Gly-Phe-Leu), as visualized with ninhydrin reaction, werescraped from the plate, and the incorporated radioactivity was countedin a liquid scintillation counter. A comparison of the amount of ³H-labelled Try-Gly-Gly formed by uninhibited enzyme with that formed inthe presence of various concentrations of an inhibitory compound allowscalculation of the I₅₀ value, i.e., the concentration of test compoundproducing 50% inhibition of the enzyme under the conditions describedabove.

    ______________________________________                                        Compound             IC.sub.50 nM                                             ______________________________________                                        N--(DL-3-Mercapto-2-methyl-                                                                        150                                                      1-oxopropyl)-L-arginine                                                       (Ex. 1)                                                                       N--[DL-2-(Mercaptomethyl)-                                                                         85                                                       1-oxo-3-phenylpropyl]-L-                                                      arginine (Ex. 2)                                                              N--(DL-3-Mercapto-2-methyl-                                                                         5                                                       1-oxopropyl)-L-tryptophan                                                     (Ex. 3)                                                                       N--(DL-3-Mercapto-2-methyl-                                                                        2.5                                                      1-oxopropyl)-L-tyrosine                                                       (Ex. 4)                                                                       3-Hydroxy-N--(D,L-3-mercapto-                                                                       5                                                       2-methyl-1-oxopropyl)-L-                                                      tyrosine (Ex. 5)                                                              3-Hydroxy-N--(D-3-mercapto-                                                                        2.5                                                      2-methyl-1-oxopropyl)-L-                                                      tyrosine (Ex. 6)                                                              (±)-N.sup.2 --[2-(Mercaptomethyl)-4-                                                            30                                                       methyl-1-oxopentyl]-L-                                                        arginine (Ex. 7)                                                              (±)-N--[2-(Mercaptomethyl)-1-                                                                    3                                                       oxo-3-phenylpropyl]-L-leucine,                                                ammonium salt (Ex. 8)                                                         ______________________________________                                    

EXAMPLE 24

1000 tablets each containing 100 mg. of3-hydroxy-N-(D-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine are producedfrom the following ingredients:

    ______________________________________                                        3-Hydroxy-N--(D-3-mercapto-2-methyl-                                                                   100    g.                                            1-oxopropyl)-L-tyrosine                                                       Corn starch              50     g.                                            Gelatin                  7.5    g.                                            Avicel (Microcrystalline cellulose)                                                                    25     g.                                            Magnesium stearate       2.5    g.                                            ______________________________________                                    

The 3-hydroxy-N-(D-3-mercapto-2-methyl-1-oxopropyl)-L-tyrosine and cornstarch are admixed with an aqueous solution of the gelatin. The mixtureis dried and ground to a fine powder. The Avicel and then the magnesiumstearate are admixed with the granulation. This is then compressed in atablet press to form 1000 tablets each containing 100 mg. of activeingredient.

The compounds of Examples 1 to 5 and 7 to 22 can be formulated in asimilar manner.

What is claimed is:
 1. A method of inhibiting the degradation ofenkephalins by the enkephalinase enzyme in a mammalian host whichcomprises administering to said mammal an enkephalinase inhibitingeffective amount of the enkephalinase inhibitor of the formula ##STR50##or a pharmaceutically acceptable salt thereof wherein: R₁ is straight orbranched chain alkyl of 1 to 4 carbons, benzyl or phenethyl;R₂ is##STR51## and n is an integer from 1 to
 4. 2. A method of claim 1wherein R₁ is --CH₃, --CH₂ --CH(CH₃)₂ or ##STR52## and n is one.
 3. Themethod of claim 2 wherein R₁ is --CH₃ and R₂ is ##STR53##